This invention relates to angiostatin, an inhibitor of angiogenesis.
Angiostatin, a proteolytic fragment of plasminogen believed to consist of kringles 1 through 3 and all or part of kringle 4 is a potent inhibitor of angiogenesis and the growth of tumor cell metastases. O""Reilly et al., Cell, 79, 315-328 (1994); PCT application WO 95/29242. Angiostatin is found in vivo in tumor-bearing mice. O""Reilly et al., Cell, 79, 315-328 (1994); O""Reilly et al., Nature Med. 2, 689-692 (1996). The enzymatic mechanism by which angiostatin is generated in vivo remains unknown.
Angiostatin activity can be generated in vitro by limited elastase proteolysis of plasminogen. Sottrup-Jensen et al. in Progress in Chemical Fibrinolysis and Thrombolysis, 3, 191-209 (Davidson et al. eds. 1978). A recent abstract proposes that angiostatin is generated by macrophages infiltrating primary tumors and releasing elastase activity, which then cleaves plasminogen to form a protein having angiostatin activity. Dong et al., Proc. Am. Assoc. Cancer Res., 37 58 (1996). However, while limited elastase cleavage of plasminogen will yield a fragment or fragments having angiostatin activity, elastase will further digest the fragment(s) to inactive peptides, and therefore, is probably not the enzyme that generates angiostatin in vivo.
As noted above, angiostatin may be generated in vitro by limited elastase proteolysis of plasminogen. This method has several disadvantages. First, while elastase cleaves plasminogen to generate a fragment containing kringles 1-3, it is not known if this cleavage is at the normal sites where cleavage occurs to produce angiostatin in vivo. Therefore, the elastase-derived angiostatin may have altered in vivo processing with altered activity in humans. It may also be immunogenic if the sites of peptide cleavage are different from normal angiostatin.
A second means of producing angiostatin is by expressing the desired kringle domains of the plasminogen cDNA or gene in an expression vector in prokaryotic or eukaryotic cells. See PCT application WO 95/29242. This approach is also limited since the appropriate domains to express are not known. The product may also be immunogenic and may not be processed in humans as would be the product generated by cleavage of plasminogen by the normal in vivo enzymes.
Finally, angiostatin can be isolated from the body fluids of animals in which it is produced. See PCT application WO 95/29242. However, angiostatin cannot be produced in sufficient quantities for disease treatment in this manner, and the angiostatin may be contaminated with infectious agents when isolated from such sources.
Clearly a need exists for a method of producing native angiostatin in large quantities. xe2x80x9cNative angiostatinxe2x80x9d is defined herein to be the angiostatin produced in vivo or angiostatin, no matter how produced, which is the same as the angiostatin produced in vivo.
The present invention provides such methods. These methods are based on the discovery that a conditioned culture medium (CCM) produced by culturing cancer cells, primary endothelial cells, smooth muscle cells or fibroblasts produces angiostatin when contacted with plasminogen or plasmin. The active factors in the CCM have been identified to be a plasminogen activator and a sulfhydryl donor. Thus, the angiostatin produced by the use of a plasminogen activator and sulfhydryl donor is the same as angiostatin produced in vivo, i.e., it is native angiostatin.
In one method of the invention for producing angiostatin in vitro, plasmin is contacted with a sulfhydryl donor to produce the angiostatin. The plasmin may be generated by contacting plasminogen with a plasminogen activator. Most conveniently, all of the reactants (plasminogen, plasminogen activator and sulfhydryl donor) can be contacted simultaneously to produce the angiostatin.
The angiostatin produced by this method, along with any remaining reactants, or angiostatin purified or partially purified from the reactants, may be administered to an animal, including a human, in need thereof. Animals in need of angiostatin are animals suffering from an angiogenic disease.
The invention further provides a composition for generating angiostatin. The composition comprises a sulfhydryl donor and a plasminogen activator. Two embodiments of the composition are CCM produced by culturing cells capable of producing plasminogen activator and a lysate of such cells.
The invention also provides a method of treating an angiogenic disease comprising administering to an animal suffering from such a disease an amount of a sulfhydryl donor effective to cause the conversion of plasmin to angiostatin. The plasmin may be that produced by endogenous plasminogen activator(s) from endogenous plasminogen. Alternatively, the method may further comprise administering an effective amount of plasmin. In yet other embodiments, a plasminogen activator may be administered to the animal to produce the plasmin from endogenous plasminogen or from an effective amount of administered plasminogen.
The invention further provides a container holding a plasminogen activator, alone or in combination with sulfhydryl donor. The container has a label thereon instructing administration of the plasminogen activator or the combination of the plasminogen activator and sulfhydryl donor to an animal suffering from an angiogenic disease. The invention also provides a container holding a sulfhydryl donor with a label thereon instructing administration of the sulfhydryl donor in an amount effective to cause the conversion of plasmin to angiostatin.
The invention also provides a protein having the following characteristics: (a) it is a fragment of plasminogen; (b) its N-terminal amino acid is the same as the N-terminal amino acid of plasmin; (c) its C-terminal amino acid is in kringle 5; and (d) it inhibits angiogenesis. In one embodiment, the protein is native angiostatin. The invention further provides a DNA molecule coding for the protein, the DNA molecule operatively linked to expression control sequences, a host cell comprising the DNA molecule operatively linked to expression control sequences, and a method of producing the protein comprising culturing the host cell. The protein may be used to treat angiogenic diseases by administering an effective amount of the protein to an animal suffering from such a disease. An animal suffering from such a disease may also be treated by administering to it a transgene coding for the protein. Preferably, the protein coded for by the transgene is native angiostatin.
Finally, the invention provides an antibody which binds selectively to the protein. Such an antibody may be used to purify the protein from materials containing it. Also, such an antibody which binds selectively to native angiostatin may be used in methods and kits to detect or quantitate native angiostatin.